One of the host defenses against HIV1 is “APOBEC3G” and related proteins. These proteins force HIV to hypermutate, killing its ability to replicate in the next cell it infects. On the other hand, it looks as if low-level mutation by APOBEC3G over the decades has driven HIV evolution:

We have found that hA3G activity acting on prior generations of virus has left detectable footprints in the HIV-1 genome. 2

HIV can only infect human cells because it has a defense against APOBECs: the viral protein “vif” causes APOBECs to be destroyed, and the virus is able to replicate without being hypermutated.

So let’s say we develop an antiviral drug that blocks vif. APOBECs would drive hypermutation of the virus. This hypermutation would include the gene encoding vif. Would this mutation, in a kind of molecular judo, drive rapid evolution of vif, so that it becomes resistant to the drug?

According to some experiments 2 both in cells and in silico, perhaps not:

However, since the predicted effect on resistance to standard antiviral drugs is likely to be small, we propose that concerns over increased resistance mutations should not impede development of HIV-1 Vif as a candidate drug target. 2

I’m not quite convinced this paper was actually modeling the phenomenon they say they’re modeling — is vif that’s shut off by deliberate mutations the same as vif that’s blocked by a (hypothetical) drug? But it’s a useful start, anyway.

We know that the immune system can destroy tumors. We also know, unfortunately, that by the time we see a tumor, immunity probably won’t destroy the tumor. There are lots of reasons for that. One is that tumors are essentially part of the normal body, so it’s normal for the immune system to ignore them. It looks as if you need to have immunity that’s just right to get rid of a tumor.

Tumors arise from normal self cells,1 that the immune response has been programmed to ignore. Now, the process of becoming a tumor is not normal, and so tumors are not entirely normal self any more — meaning that there are likely to be some targets in most if not all tumors. But in all but the most reckless tumors the differences between abnormal and normal are relatively small, compared to, say, a virus-infected cell that contains many potential targets.

There’s actually a long list of known tumor antigens; the T-cell tumor peptide database lists many hundreds of them. But most are not truly specific for the tumor. The’re actually normal self antigens; they’re derived from proteins that are overexpressed in tumors, or that are differentiation antigens or “cancer-germline” antigens that are normally also found in self tissues. What’s more, these normal self antigens are the most interesting tumor antigens, as far as clinical utility is concerned. Mutations can make brand-new, non-self targets for the immune system, but they’re going to be sporadic targets, often unique to individual tumors — not something you can prepare for. The normal antigens, though, are likely to be predictable, common targets; it’s conceivable that tumor vaccines can be prepared in advance.

Human melanoma cell

If these antigens were common (which they are, in some tumor types — like melanoma), and they were good targets for the immune system, then we wouldn’t see much cancer. We do see melanomas quite often, and part of the reason may be that the immune system generally responds quite weakly to these antigens. Why is that? And, more to the point, how can we make the immune system respond more strongly? A recent paper in the Journal of Experimental Medicine2 offers answers for both of these questions.

From work in the past couple of years, we now have decent estimates of how many T cells there are that can react with any particular target. (See here and here for my discussion of the earlier papers.) A reasonably strong immune response to a non-self epitope might originate from maybe 100 or so precursor T cells. There’s a rather wide range of frequency for these precursor cells, say from 20 to 1000; and to some extent, the fewer T cells there are the weaker (the less immunodominant) the immune response.

We expect T cells against normal self targets to be less common, because they should be eliminated as they mature in the thymus. Some may survive, though, and we would count on these survivors to attack the normal (albeit overexpressed, or abnormally present) target in the cancer cells. But just how rare are they?

Rizzuto et al say they’re really rare (this was in mice, by the way); at least ten times less abundant than T cells against non-self antigens. If you look at the range I gave for “normal” precursors, that could mean there are fewer than 5 or 10 precursors. If the average is “fewer than five”, then quite possibly some mice have only two, or one, or no precursors. You can’t have much of a response with no precursors.

So there’s a weak anti-tumor response because there aren’t many T cells in the body that can respond to the normal self targets in the tumor. That’s not really a surprise, but it does raise the question, What if there were more of the T cells? To ask that question, Rizzuto et al. tried transferring more of these precursor T cells into tumor-bearing mice — starting at around the normal level for a precursor to non-self antigen, and going up from there — and then vaccinating with the appropriate target.

The effects were pretty dramatic. With no supplemental T cells (that is, with the natural, very low, level of T cell precursors) the mice all died of the tumor quickly. At the middle of the range, almost all of the mice rejected the tumor. And at the highest levels of transfers? The mice all died again. Having enough T cells to respond was protective, but putting in too many made them useless.

These results identify vaccine-specific CD8+ precursor frequency as a remarkably significant predictor of treatment and side-effect outcome. Paradoxically, above a certain threshold there is an inverse relationship between pmel-1 clonal frequency and vaccine-induced tumor rejection.2

Mouse melanoma cell

(My emphasis) This paradoxical effect is probably because the numerous T cells started to compete with each other so that none of them were properly activated; they only saw effective-looking polyfunctional T cells at the lower transfer levels.

In other words, if you’re going to transfer T cells to try to eliminate a tumor, more is not necessarily better. Quality and quantity are both important factors, and quantity helps determine quality.

One question I have is how this relates to tumor immune evasion. Many tumor types acquire mutations, as they develop, that block presentation of antigen to T cells. Are these mutations perhaps only partially effective — giving the tumors sufficient protection against the tiny handful of natural precursors they “expect” to deal with, but not against a larger attack after, say, vaccination — or are they more complete, and protective even if the optimal number of T cells are transfered? I’d guess that it would depend on the tumor, but it looks as if it might be a relevant question and it would be nice to have more than a guess.

Our results show that combining lymphodepletion with physiologically relevant numbers of naive tumor-specific CD8+ cells and in vivo administration of an effective vaccine generates a high-quality, antitumor response in mice. This approach requires strikingly low numbers of naive tumor-specific cells, making it a new and truly potent treatment strategy. 2

Something that’s puzzled me for years is why the same kinds of tumors tend to have the same kinds of immune evasion mechanisms. And I’m not going to give an answer, just trying to share the confusion a little.

What I mean is this:

It has been demonstrated that human tumors of distinct histology express low or downregulated MHC class I surface antigens … The distinct frequency of MHC class I abnormalities is caused by total HLA class I antigen loss, HLA class I down-regulation as well as loss or down-regulation of HLA class I allo-specificities. However, the frequency and mode of these defects significantly varied between the types of tumors analysed and could be associated in some cases with microsatellite instability. 2

(My emphasis) As I’ve noted here several times (most specifically here) tumors very often evade the immune system as they mature. Cytotoxic T lymphocytes (CTL) can control tumors in the tumors’ eary stages, but by the time we detect a tumor clinically the tumor is almost always resistant to the immune system. They do this in various ways, including inducing regulatory T cells, but also by mutating themselves to make themselves invisible to CTL (and other components of the immune system, but let’s keep it simpler for the moment).

There are a myriad ways for a tumor to become invisible, at the molecular level. The MHC class I antigen presentation pathway is long and complex, and for any partiuclar tumor there are likely to be many different bottlenecks, points of attack. Since tumors are all independent events3, so at first, and even second, glance, there’s no obvious reason why tumors of the same type should find a similar approach. That is, just because two colon carcinomas look the same histologically in two different individuals, there’s no link between them. 4 Why should colon carcinomas avoid CTL using one set of mutations, while, say, breast cancers use a different set of mutations? Yet apparently, that’s what tends to happen; for example:

Mutations or deletions in β2-m were detected in colon carcinoma (21%), melanoma (15%) and other tumors (<5%). So far, no mutations in β2-m have been found in RCC lesions, bladder and laryngeal tumors despite MHC class I loss or downregulation. … haplotype loss was found in head and neck squamous cell carcinoma (HNSCC) with a frequency of 36%, whereas in renal cell carcinoma (RCC) LOH only occurs in approximately 12% of tumor lesions analyzed. 2

If we saw these patterns only with virus-associated cancers, such as cervical carcinomas and even hepatic carcinomas, there would at least be a common link, but these tumors are not (as far as we know) caused by viruses in humans.

Part of the answer may be that the particular oncogenes associated with different tumor types lead to particular transcriptional hot-spots, and being a transcriptional hot-spot makes the region a mutational hot-spot as well, but at least as I understand it that’s not enough to account for the trends.

barring such weird things as canine transmissible venereal tumor and Tasmanian Devil facial tumors; see here for more on those[↩]

The comparison is, of course, viruses. A herpesvirus of chickens, and one of humans, may both use immune evasion mechanisms, but they have a common ancestor even if it’s a couple of hundred million years ago.[↩]

It’s well known that HIV mutates rapidly in infected patients in order to escape from the immune system. The mutations in HIV track with the peptides that bind to MHC class I in any particular patient. When the virus is transmitted to a new patient, though, those mutations don’t help it much, because MHC is so variable between individuals that the new infected person will very likely have a different MHC class I pattern. (In fact, the mutations the virus developed in the first patient, are likely to be actively harmful to the virus.) The virus has to start all over again and discover a new path toward immune escape. Over a long enough time, the virus may be able to slowly accumulate mutations that allow it to escape from the worst of the MHC class I alleles (see here for a possible example), but it’s very difficult, simply because MHC is so diverse.

But MHC class I itself is only the final stage of a longish pathway of antigen presentation — the route by which peptides are produced, modified, transferred into the right location, bind to the right proteins, all that stuff. (If it’s slipped your memory a little, I made a summary page for MHC class I antigen presentation here.) Within that pathway, at least in humans, it’s only the MHC class I heavy chain itself that’s wildly diverse; the other steps are pretty similar between any two individuals. So why doesn’t the virus mutate to avoid one of these monomorphic steps, and then not have to worry about re-mutating all over again after the next transmission?

Putting that less teleologically, why don’t mutations in HIV, that allow it to escape from the monomorphic steps in antigen presentation, persist in each new individual and accumulate within the population? Those mutations should be just as beneficial to the virus in the new infected person as in the original infectee.

(My emphasis) And the reason is the same reason other immune escape mutations don’t easily accumulate in the population: MHC is too diverse. If I follow the argument correctly, because the other components of the system are monomorphic, they have a very broad specificity for peptides, whereas MHC itself has a fine specificity. The virus can’t mutate every possible sequence in its genome that would interact with, say, TAP, because there would be thousands of them. If a mutation that prevents TAP binding does arise in one host, it’s selected because it prevents recognition of a particular MHC class I-binding peptide, and when it moves into a new host that peptide is no longer relevant for immune escape, so it’s not selected any more.

That means that, even taking the whole antigen presentation pathway into account:

The total number of predicted epitope precursors and CTL epitopes in a large population data set of HIV-1 clade B sequences is not decreasing over time. 1

I am a little cautious about accepting this paper completely, because it’s heavily based on database analysis without a lot of testing; we don’t actually know whether the escape mutations they identify for TAP actually do escape TAP, for example. They make a number of arguments, in passing, for the accuracy of the epitope prediction programs out there; I am slowly backing in to some acceptance of the notion that the predictive programs are getting pretty good, which wasn’t my position a couple of years ago, but I still am not convinced they’re as good as they say here.

But the conclusion is fairly simple and straightforward, and it leads to an interesting suggestion:

… we speculate that only one of the steps in the antigen presentation pathway has to be polymorphic to prevent pathogens from adapting to any step in the pathway. The mechanism functions best when the polymorphy occurs at the most specific step in the pathway, as that increases the fraction of epitope precursors that is not under selection pressure. While in humans it is the MHC class I molecules that are highly polymorphic and specific, other solutions do appear to exist. The TAP molecules of rats are more specific than the human TAP, and have a limited functional polymorphism, and the TAP and MHC genes of chickens are equally polymorphic on the nucleotide level 1

Chicken MHC is an interesting case, and is very strongly linked to resistance to some pathogens. But the reason for the tight linkage to resistance isn’t really known; there’s no obvious reason at the level of the MHC. It might be interesting to look at TAP as part of the resistance, as well. I have some chicken stuff in the lab, and I should see if we can test that.

Schmid, B., Kesmir, C., & de Boer, R. (2008). The Specificity and Polymorphism of the MHC Class I Prevents the Global Adaptation of HIV-1 to the Monomorphic Proteasome and TAP PLoS ONE, 3 (10) DOI: 10.1371/journal.pone.0003525[↩][↩][↩][↩]

Any time a species meets some kind of barrier, there’s going to be selection to overcome that barrier. In the case of pathogens, one major barrier they have to hurdle is their hosts’ immune systems. What’s more, this isn’t a simple, static barrier. Immune systems change on a day-to-day basis; and immune systems also change on a population basis, as the individuals in the host population are in turn selected by the pathogen.

Last week I talked about an example where a population — frogs in the UK, in this case — are apparently being selected by a pathogen. The relatively recent introduction of frog virus 3 into the UK has caused large-scale die-offs of frogs there, and Teacher et al.1 have just shown evidence that the survivors have been selected for a particular MHC class I type.

MHC class I is often associated with resistance to viruses, because it’s responsible for recognition by antiviral T cells. What probably happens is that viruses sweep through a population, infecting (and imposing a selective pressure on) most members of the population. A few individuals that happens to have some particular MHC class I type are relatively resistant to the virus, and have a selective advantage; that MHC allele becomes more frequent in the population; and the population as a whole becomes relatively resistant to the virus. Of course, this now presents a new barrier to the original virus, and there’s selective pressure on it; virus mutants that are resistant to that particular MHC type do better; the virus sweeps through the new population; and a new minority with a different MHC type has a new selective advantage.

This is the most popular model (“frequency-dependent selection”), but it’s been hard to definitively show examples of it because things are happening on a evolutionary timescale. Even with the very rapid (as evolution goes) change in the UK frogs’ MHC, we don’t have all the pieces. We see that frogs in the UK have a different set of MHC alleles than those frogs that haven’t been exposed to FV3, but we don’t have the population frequencies of these alleles over the time since the virus was introduced. And we don’t have examples of the virus accommodating itself to the new MHC; we’d see that as virus sequences changing over time.

Last week I ended the frog story by saying:

Some people may wonder if this frog virus story has any real relevance to humans. Well, apart from the pure scientific interest of tracking a potential frequency-dependent selection event in real time, one of the clearest links between an MHC class I allele and resistance to a viral infection is in humans, where the MHC class I alleles HLA-B27 and HLA-B57 are linked to resistance to HIV and HCV. Is it possible for HIV to adapt at the population level, so that the dominant strains of HIV in the world are no longer contained by HLA-B57? More generally, if we succeed in developing a T cell-based vaccine against HIV, it will probably have strong allele-dependent effects — will HIV adapt to this vaccine?

Astute readers2 may have guessed that I wasn’t just guessing wildly, and indeed I had already seen the paper from Kawashima et al.,3 on exactly this topic.

Even though HIV is generally incredibly good at ripping through human immune responses without being controlled, there are some people who are long-term non-progressors (LTNP); they’re infected with HIV, yet they manage to control the virus pretty well, without antiviral treatment, for long periods. Many of these people, it turns out, have a particular subset of MHC class I types; they’re much more likely than the general population to have the HLA-B51, HLA-B57, or HLA-B27 MHC class I alleles.

HIV normally mutates very rapidly within infected individuals, so that as an antiviral immune response arises the virus may be temporarily controlled, but the new mutations that arise escape from the immune control and continue to replicate. It seems that this immune escape is less likely to happen when the individual has one of the LTNP-associated alleles, and that’s probably because the immune target associated with HLA-B51 (etc) is essential for the virus’s survival. When HIV mutates the immune target, the virus can’t replicate properly. The only way HIV can escape immune control by people with these MHC alleles is to make multiple mutations at the same time, compensating for the escape mutation with several other changes. These multiple mutations are exponentially less probable than single mutations, so the virus is essentially controlled, for a long time.

Humans are today a very large, highly mixed population, and it would take a vast plague, even worse than HIV, to rapidly cause frequency changes that we could measure in the brief period since HIV become common.4 But that hasn’t always been true; humans historically have included relatively small and isolated populations subject to intense disease selection, and we believe we see the outcome of that today in that different human populations have different frequencies of HLA-B51, B57, and B27 — the equivalent of frogs in the UK vs. elsewhere.

What’s happening to HIV in those areas where HLA-B51 is common? The prediction is that viruses that have managed to make the mutations that give resistance to HLA-B51 should have a selective advantage in those areas that isn’t seen elsewhere. That’s precisely what Kawashima et al. saw.

… the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. 3

HLA-B51 complexed with an immunodominant HIV peptide

Immune escape isn’t the only selective pressure on HIV. There’s the ability to spread from one individual to another, for example, which isn’t necessarily linked to immune escape. In principle, some of the other selective factors may counteract immune escape selection. And in general, some (though not all) of the mutations that allow a virus to escape immune control by on individual are harmful to the virus. That means that some of the escape sequences will quickly revert back to the generic HIV sequence. If HLA-B51 is rare in the population, the virus will constantly be reverting back to generic sequences and the HLA-B51-resistant strain will not particularly accumulate. But if HLA-B51 is common, even these reverting sequences will build up in the population.

As anticipated, non-reverting variants such as I135X accumulate at the population level, but even rapidly reverting mutations such as T242N can accumulate, if the selection rate exceeds the reversion rate 3

Perhaps as a result, formerly-protective MHC alleles are no longer protective in some areas:

Data here suggest that, whereas 25 years ago HLA-B*51 was protective in Japan, this is no longer the case. The apparent increase in I135X5 frequency in Japan over this time supports the notion that HLA-B*51 protection against HIV disease progression hinges on availability of the HLA-B*51-restricted TAFTIPSI6 response. However, whether this is the case remains unknown. 3

Any effective anti-HIV vaccine will probably rely on antiviral T cells, and will therefore rely on MHC class I presentation. What this paper suggests is that HIV is likely to be a moving target. Even if an effective vaccine is developed, it is possible that the virus will gradually evolve resistance to the vaccine.

Thus, the data presented here, showing evidence that the virus is adapting to CD8+ T-cell responses, … highlight the dynamic nature of the challenge for an HIV vaccine. … The induction of broad Gag-specific CD8+ T-cell responses may be a successful vaccine strategy, but such a vaccine will be most effective if tailored to the viral sequences prevailing, and thus may need to be modified periodically to keep pace with the evolving virus. 3

Since we still don’t have any vaccine that protects against HIV at all, this is pretty much a hypothetical worry. Still, it’s something to think about for the future.

Although there are quite a few viruses that infect and then persist in the infected animal for a long time, most of these viruses don’t cause a lot of problems during the persistent state. Herpesviruses, adenoviruses, and several other families can stick around for a long time (life-long, in the case of most herpesviruses) and although you’ll sometimes see occasional recurrence, and occasionally there can be serious disease from the reactivation (examples being shingles, from recurrent varicella-zoster virus, or the very rare cancers associated with Epstein-Barr virus) — for the most part these are really unusual outcomes. Mostly we can stroll around with our complement of viral passengers and we’re perfectly fine with it.

There are a handful of exceptions, in humans and in other animals, where viruses that cause chronic infection also cause chronic, severe disease. With these viruses we’d like to know why they become chronic in the first place (why doesn’t the immune system eliminate them?), and we’d like to know why they cause disease (why aren’t they like our friendly neighborhood herpesvirus)? We don’t have good answers for either of these questions. In fact, I don’t think we even have a good sense if the answers are the same for different viruses, or whether each of them manages to persist and cause chronic disease through their own unique factors.

Two of the most prominent chronic virus diseases in humans are HIV and HCV (hepatitis C virus). A recent paper1 suggests that these guys may have at least something in common in the way they escape immune control, and in the way the immune system controls them.

It’s pretty well known that one way HIV escapes immune control is that it mutates so fast, the immune system can’t get a grip on it. In particular, the cytotoxic T lymphocytes (CTL) that are specialized to control virus infections need to recognize a stretch of about 9 amino acids (a peptide, in other words), and if that sequence changes the CTL may no longer recognize the virus. (I’ve talked about that here and here.) It’s also become clear that HCV does the same thing, although perhaps less dramatically than does HIV, and that this mutational immune escape is one reason HCV can persist and continue to cause disease (see here for more).

With HIV, there are a number of “elite controllers” who seem to be resistant to the virus’s ability to mutate away from immune control. In many cases this seems to be because the CTL in those individuals are focused on a particular critical stretch of amino acids that simply can’t mutate without severely damaging the virus (reducing HIV’s replicative fitness). The reason these elite controllers select that critical peptide is that they have a MHC class I allele that specifically binds to that peptide sequence. The HLA-B27 and HLA-B57 alleles seem to be particularly likely to find critical peptides, and people with those MHC alleles are more likely to be elite controllers.

The new HCV paper1 shows that HLA-B27 is also protective against HCV infection 2, and for the same reason — the HCV peptide that binds to HLA-B27 is a critical sequence that can’t mutate much without severely damaging the virus. (When HCV does mutate away from HLA-B27-mediated control, it’s because it has developed multiple mutations in the peptide, not just one, and it’s exponentially3 harder for the virus to make two mutations vs. one.)

Is it just a coincidence that HLA-B27 is involved in both cases, or is there something specially magical about HLA-B27? I’d be inclined to say it’s just coincidence except that HLA-B27 is such a special molecule4 already. It’s involved in all kinds of disease risks, both reducing the risk of some infectious diseases like HIV and HCV and dramatically increasing the risk of autoimmune diseases like ankylosing spondylitis and many others. And off the top of my head, I think it’s one of the very ancient and highly diversified groups of HLA molecules. So maybe there is something about it that manages to focus on critical viral peptides, or that makes it a particularly strong stimulator of CTL, and that gives it a selective advantage that outweighs its increased risk of autoimmune disease.

I know all my regular readers1 are expecting me to talk about the bombshell announcements that NK cells have memory, but I’ll put that off for a bit and instead quickly note a very cool advance on a story I’ve mentioned a few times before.

Interferons are among the most critical early warning and protective cytokines, and they’re so effective that just about any successful virus of vertebrates has strong defenses against them. Without those defenses, the virus is essentially dead. For example, in some cases the the virus’s interferon blocker only works in one species, and that limits the virus to infecting that species only; if you get rid of the interferon response that the virus can’t deal with, then it’s perfectly capable of infecting other species.2

Influenza viruses are no exception; they possess a gene (NS1) that protects them against the host interferons. (Some strains of influenza have especially effective NS1 functions, and it’s been suggested that those with the most effective interferon blockers are the most virulent pathogens — like the 1918 flu, or avian influenza). I’ve talked about NS1 before here.

When you eliminate NS1 from influenza, the virus is — as you’d expect — greatly weakened, and infection with these defective viruses cranks up interferon and thereby, in turn, cranks up the rest of the immune response. That gives you a highly attenuated, highly immunogenic virus, which is exactly what you want to use for a vaccine; and indeed, NS1-defective influenza viruses are apparently effective and safe vaccines.3 (I’ve previously mentioned a vaccinia virus vaccine technique that follows a similar approach, with similar results.)

So NS1 is clearly an extremely important molecule for influenza, and without it, the virus is basically harmless. What if you could block NS1 after an infection? Would it do the same thing — in other words, would an NS1-blocker be an antiviral treatment?

As it turns out: Yes, it would. Daniel Engel’s lab developed a group of compounds that inhibit influenza NS1. These things inhibit influenza growth in cells, and (although NS1 has functions other than blocking interferon) the effect was dependent on the interferon response. 4

A couple of viral immune evasion molecules have already been targeted for antiviral therapy — for example, Luis Sigal has shown that a poxvirus immune evasion molecule is a good vaccine target5 — but as far as I know this is the first antiviral compound that’s specifically been developed to inactivate an immune evasion molecule, and it offers the potential for a brand-new class of antivirals. Of course there are still huge barriers between these particular compounds and actual therapy in infected animals, but it’s encouraging that they work at all, and I’m interested in seeing what arises from it.

I’ve quoted before that “the stupidest virus is smarter than the smartest virologist”. Adenoviruses are far from the stupidest viruses, and even after 55 years of study, and nearly 40,000 papers in PubMed, adenoviruses still throw surprises at us on a regular basis. Last week, while talking about herpesviruses, I added that “the other virus families that are known to evade MHC class I are human adenoviruses, which now turn out to establish true latency”. True latency has been hinted at for a while, but Linda Gooding’s group has added much more support for it in a paper in press at J Virol.1

Adenoviruses are very, very common viruses, both in humans and in many other species. In humans there are over 40 different adenovirus “species”, mostly causing cold-like symptoms and mild gastrointestinal disease. Occasionally, and perhaps with increasing frequency, there’s a more or less widespread outbreak of moderate disease, but in general these guys are not huge problems as far as mortality in immune-competent people.

As well as being common, many adenovirus species are pretty easy to grow in cultured cells, and so it’s not surprising that they were isolated and described a long time ago. In fact, the first identification of adenoviruses was as an accidental isolation in 1953, when Rowe et al noticed that the tonsil cell cultures they were growing were showing signs of viral infection.2 Since then, adenoviruses have been used as models for a huge number of cell biology functions (the Nobel on splicing came from work on adenoviruses, just as one example), as well as cancer biology and lots of other things; adenoviruses have also been used as workhorses for the viral vector field, moving into gene therapy as well.

If you look in lots of tonsils, you’ll find lots of adenovirus; something like 80% of samples turn up positive. 3 That’s much higher than the disease rate, of course, and it’s also much higher than any plausible incidence rate — that is, those can’t all be new infections, in the past week or so, that simply haven’t been cleared by the immune system. That means that adenoviruses must be able to persist for some fairly significant period — months to years — after their initial infection, without causing symptoms.

This long-term persistence by adenovirus is one of its most characteristic, um, characteristics, but it’s been completely mysterious. (If you don’t believe me, here’s what Bill Wold and Marshall Horwitz say in the latest version of the authoritative Fields Virology: “We really do not understand whether and how adenovirus persists at very low levels in humans … “)

For adenoviruses, usually the term “persistence” is used, rather than latency, because “latency” has a specific meaning: Basically, a latent virus is still present at the genome level, but isn’t capable of forming a new virus particle. Herpesviruses are the main viruses that are known to do this. Some retroviruses set up latent infection by intergrating into the host genome, but that’s a little different story. Herpesviruses can maintain their own genomes in the latently-infected cell, but in a form that’s independent of the host genome. As I’ve noted before, this latency may be tightly linked to immune evasion functions. The other option is mere “persistence”, where presumably the virus would remain in a replicating form, and would be capable of forming a new virus, perhaps at a very slow turnover rate of replication.

A handful of papers have suggested that adenoviruses might establish truly latent state4, but this new paper from Gooding’s lab1 is the most convincing I’ve seen yet. They compared infectious virus to the amount of viral DNA — that is, to viral genomes — and concluded that “only a small amount of viral DNA is present as infectious virus, even in samples with large amounts of viral DNA. … time in culture also appears to activate latent virus in the tissues, which was detected by transferring “activated” lymphocyte-derived virus onto permissive A549 cells.” (This is exactly how you detect latent infection by herpesviruses, in general — you transfer latently-infected tissue onto other cells, and over time the virus reactivates from latency to become infectious virus, and shows up on the permissive cells.) They showed that on initial exam the virus wasn’t making any transcript, again a requirement for true latency, and that over time transcripts began to appear as the virus reactivated.

As a side note, Gooding notes dryly that sloppy technique may be part of the reason adenoviruses were so often found in tonsil explants, a suggestion I hadn’t heard before.

Reports of infection of laboratory workers in the early adenovirus groups suggest that some exogenous contamination might have elevated the frequency with which live virus was found in these studies.

Finally, Gooding re-emphasizes the same point I’ve made here:

Furthermore, like all DNA viruses that form latent or persistent infections, human species C adenoviruses encode a variety of gene products, primarily within the E3 transcription unit, that function to counteract host anti-viral defense mechanisms. We have previously reported that the E3 promoter is up-regulated when cells are exposed to signals that activate T lymphocytes. Hence, it appears likely that the immune evasion strategies of these viruses are directed toward protecting the T lymphocyte from destruction during the period of viral activation from latency.

(My emphasis) As far as that goes, it’s particularly interesting to me that only human adenoviruses, and not even all of them, have MHC class I immune evasion functions. Does that mean that that particular function is less critical for latency, or does it mean that non-human adenoviruses don’t establish true latency (even in these humans, they really only found group C adenoviruses to be latent, though that may just reflect tissue preferences), or what?

Herpesviruses are unusual1 in their ability to establish a long-term latent infection in their hosts. Another unusual2 trait is that herpesviruses apparently all have genes that block MHC class I recognition by cytotoxic T lymphocytes. (I suspect that we will also find that evasion of NK cells is also universal among herpesviruses, but that’s still a new and growing field.) The other virus families that are known to evade MHC class I are human adenoviruses, which now turn out to establish true latency;3 HIV (which also has a true latent stage, though I don’t know if it’s relevant here); a handful of poxviruses, which as far as I know don’t do much in terms of latency; and some papillomaviruses4 which I don’t think have a real latent stage.

I’ve noted previously that these two unusual characteristics of herpesviruses may be linked, in that perhaps the herpesvirus MHC class I immune evasion is important for establishing latency. A review in Nature5 points out that herpesviruses are also unusual in that they tend to have a lot of micro-RNAs, and suggests a similar concept: That herpesviruses use miRNAs to evade the immune system and this is related to their ability to maintain a latent infection.

It seems possible that the presence of miRNAs in herpesviruses is associated with the characteristic ability of herpesviruses to establish long-term latent infections. Avoiding the host immune response is particularly important during latent infection, and viral miRNAs not only have the advantage of not being recognized by the host immune system but also might be an ideal tool for attenuating immune responses by downregulating the expression of key cellular genes. 5

Some viruses burn through their hosts like a flamethrower, blasting past defenses for a brief shining moment and then hurtling on through the next victim. Other viruses slip in, delicately negotiate an understanding with the immune system, and set up a long-term relationship with the host. Herpesviruses do the latter, and to me, at least, they’re more interesting than their less restrained cousins.

Herpesviruses set up long-term, usually lifelong, latent infections in their hosts. The cells that harbor the latent infections depend on the precise virus; gamma-herpesviruses like Epstein-Barr virus (EBV) typically go latent in lymphocytes. EBV only infects humans, so it’s kind of hard to do actual experiments (as opposed to observational studies) but a possible model for EBV is a mouse gamma-herpesvirus, mouse herpesvirus 68 (MHV68). I’ve talked about this virus before here, in the context of immune evasion; MHV68 has a gene that apparently allows it to avoid T cell recognition in the initial stages of infection, and this immune evasion in the early stages of infection helps determine the extent, and stability, of the latent phase. In the latent phase, though, the immune evasion molecule might not be all that helpful. What regulates the numbers of persistently-infected lymphocytes?

Immunodominance is another theme I’ve talked about several times here. Immunodominance is the phenomenon in which T cells focus their response on a small number of potential targets. A typical virus — especially a large virus, like a herpesvirus — might have hundreds or thousands of potential T cell targets (“epitopes”), according to the simpler prediction programs; but in practice, only a tiny handful of those hypothetical epitopes are actually recognized efficiently by T cells in the host. The molecular mechanisms that determine immunodominance aren’t well understood, and in many cases the importance of immunodominance isn’t clear, either. In the case of HIV, and perhaps a couple of other viruses, there’s evidence that immunodominance affects the course of infection, but there aren’t many such clear examples.

MHV68 is apparently another example. A paper from Marques et al.1 shows that in one particular mouse strain, T cell recognition of a single epitope of MHV68 is critical in determining how much latent virus hangs about in the infected mouse. Mutating that single epitope (so that it was no longer recognized by T cells) cranked up the level of latent infection dramatically.

In this case, the immunodominance is not so mysterious; there aren’t all that many genes expressed during latency (pretty much by definition) and so there aren’t many possible sources for the dominant epitope. Still, it’s surprising (to me) that a single epitope can have such a dramatic effect on the pathogenesis of the virus; I can’t think of very many instances of that. An obvious question is whether this is unique to this particular mouse strain (because mouse strains typically see different epitopes) or whether this is a general phenomenon. That’s a really hard question to answer, but the authors point at a little bit of circumstantial evidence: Apparently the gene that’s the source of the epitope shows evidence that it’s under more selection than are the neighboring genes.

So the authors’ model for this virus’s pathogenesis, as I understand it, would be something like this: In the initial infection, the virus expresses many genes, and probably has many targets for the immune system. But some of the genes it expresses are immune evasion genes, which dampen but don’t eliminate the immune response, allowing the virus to get access to the lymphocytes it needs for latent infection. The virus constantly, though slowly, reactivates, so that there should be fewer latently-infected lymphocytes over time; that’s counteracted by the virus forcing the lymphocytes to replicate themselves at about the same rate. At the same time, though, latently-infected lymphocytes will be also gradually depleted by the immune system. This immune-mediated depletion depends on the virus having a target for the T cells to see; because there are only a small number of latently-expressed genes, there are only a few possible targets for the T cells. Immunodominance (probably) narrows this even more, in this case to a single T cell epitope. Get rid of that epitope and there’s no target at all, so that instead of merely keeping pace with the depletion, the virus can at least temporarily get ahead of the depletion, increasing the latent set-point of the virus.

In the case of EBV, at least, it’s likely that the latent set-point is important in disease, so (if MHV68 really is a model for EBV infection, which is a little controversial) this may be an example of immunodominance determining disease.